Cytidine deaminase (CDA) is an important enzyme in the regulation of pyrimidine metabolism, catalysing the hydrolytic deamination of cytidine to uridine and ammonia. It is one of several deaminases responsible for maintaining the cellular pyrimidine pool.Traditional methods for the estimation of CDA activity measure the ammonia production. However, the low activity of the enzyme requires long incubation times, which in turn increase the possibility of contamination by exogenous ammonia. An analytical method utilizing high-performance liquid chromatography � ultra-violet detector (HPLC-UV) was reported to determine CDA activity by measuring the uridine produced by the action of the enzyme. However, due to the lower sensitivity of the UV detector, a larger sample volume was required.Using a Shimadzu HPLC and an AB SCIEX TripleTOF 5600 mass spectrometer with a DuoSpray ion source, we introduce a more sensitive liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method to determine human plasma CDA activity based on the concentration measurement of uridine. The high mass resolution/accuracy of the mass spectrometer provided a novel quantitation approach targeting either ionized uridine molecular ion, or its fragmentation ions, with less interference from biometrics. The chromatography was performed through an Atlantis T3 column with a fast eluting gradient. The optimum enzyme substrate concentration and the enzymatic reaction time were also investigated.A LC-ESI-MS/MS method to determine human plasma CDA activity was successfully developed. By optimizing the incubation conditions and if necessary the LC-ESI-MS/MS conditions, it is possible to use such methods for measuring the activity of other enzymes of nucleic acid metabolism.

Determination of human plasma cytidine deaminase activity by a hybrid quadrupole time-of-flight tandem mass spectrometer

246th National Meeting (2013)

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